Inducible NOS inhibition reverses tobacco-smoke-induced emphysema and pulmonary hypertension in mice

Chronic obstructive pulmonary disease (COPD) is one of the most common causes of death worldwide. We report in an emphysema model of mice chronically exposed to tobacco smoke that pulmonary vascular dysfunction, vascular remodeling, and pulmonary hypertension (PH) precede development of alveolar destruction. We provide evidence for a causative role of inducible nitric oxide synthase (iNOS) and peroxynitrite in this context. Mice lacking iNOS were protected against emphysema and PH. Treatment of wild-type mice with the iNOS inhibitor N(6)-(1-iminoethyl)-L-lysine (L-NIL) prevented structural and functional alterations of both the lung vasculature and alveoli and also reversed established disease. In chimeric mice lacking iNOS in bone marrow (BM)-derived cells, PH was dependent on iNOS from BM-derived cells, whereas emphysema development was dependent on iNOS from non-BM-derived cells. Similar regulatory and structural alterations as seen in mouse lungs were found in lung tissue from humans with end-stage COPD.     

Therapeutic efficacy of TBC3711 in monocrotaline-induced pulmonary hypertension

Background

Modified function of immune cells in nasal secretions may play a role in the enhanced susceptibility to respiratory viruses that is seen in smokers. Innate immune cells in nasal secretions have largely been characterized by cellular differentials using morphologic criteria alone, which have successfully identified neutrophils as a significant cell population within nasal lavage fluid (NLF) cells. However, flow cytometry may be a superior method to fully characterize NLF immune cells. We therefore characterized immune cells in NLF by flow cytometry, determined the effects of live attenuated influenza virus (LAIV) on NLF and peripheral blood immune cells, and compared responses in samples obtained from smokers and nonsmokers.

Methods

In a prospective observational study, we characterized immune cells in NLF of nonsmokers at baseline using flow cytometry and immunohistochemistry. Nonsmokers and smokers were inoculated with LAIV on day 0 and serial nasal lavages were collected on days 1-4 and day 9 post-LAIV. LAIV-induced changes of NLF cells were characterized using flow cytometry. Cell-free NLF was analyzed for immune mediators by bioassay. Peripheral blood natural killer (NK) cells from nonsmokers and smokers at baseline were stimulated in vitro with LAIV followed by flow cytometric and mediator analyses.

Results

CD45(+)CD56(-)CD16(+) neutrophils and CD45(+)CD56(+) NK cells comprised median 4.62% (range 0.33-14.52) and 23.27% (18.29-33.97), respectively, of non-squamous NLF cells in nonsmokers at baseline. LAIV did not induce changes in total NK cell or neutrophil percentages in either nonsmokers or smokers. Following LAIV inoculation, CD16(+) NK cell percentages and granzyme B levels increased in nonsmokers, and these effects were suppressed in smokers. LAIV inoculation enhanced expression of activating receptor NKG2D and chemokine receptor CXCR3 on peripheral blood NK cells from both nonsmokers and smokers in vitro but did not induce changes in CD16(+) NK cells or granzyme B activity in either group.

Conclusions

These data are the first to identify NK cells as a major immune cell type in the NLF cell population and demonstrate that mucosal NK cell cytotoxic function is suppressed in smokers following LAIV. Altered NK cell function in smokers suggests a potential mechanism that may enhance susceptibility to respiratory viruses.     

Detection of reactive oxygen species in isolated, perfused lungs by electron spin resonance spectroscopy

Background

Interleukin (IL)-9 is a Th2-derived cytokine with pleiotropic biological effects, which recently has been proposed as a candidate gene for asthma and allergy. We aimed to evaluate the therapeutic effect of a neutralizing anti-IL-9 antibody in a mouse model of airway eosinophilic inflammation and compared any such effect with anti-IL-5 treatment.

Methods

OVA-sensitized Balb/c mice were intraperitoneally pretreated with a single dose (100 μg) of an anti-mouse IL-9 monoclonal antibody (clone D9302C12) or its vehicle. A third group was given 50 μg of a monoclonal anti-mouse IL-5 antibody (TRFK-5) or its vehicle. Animals were subsequently exposed to OVA on five days via airways. Newly produced eosinophils were labelled using 5-bromo-2'-deoxyuridine (BrdU). BrdU⁺ eosinophils and CD34⁺ cell numbers were examined by immunocytochemistry. After culture and stimulation with OVA or PMA+IC, intracellular staining of IL-9 in bone marrow cells from OVA-exposed animals was measured by Flow Cytometry. The Mann-Whitney U-test was used to determine significant differences between groups.

Results

Anti-IL-9 significantly reduced bone marrow eosinophilia, primarily by decrease of newly produced (BrdU⁺) and mature eosinophils. Anti-IL-9 treatment also reduced blood neutrophil counts, but did not affect BAL neutrophils. Anti-IL-5 was able to reduce eosinophil numbers in all tissue compartments, as well as BrdU⁺ eosinophils and CD34⁺ progenitor cells, and in all instances to a greater extent than anti-IL-9. Also, FACS analysis showed that IL-9 is over-expressed in bone marrow CD4⁺ cells after allergen exposure.

Conclusions

Our data shows that a single dose of a neutralizing IL-9 antibody is not sufficient to reduce allergen-induced influx of newly produced cells from bone marrow to airways. However, in response to allergen, bone marrow cells over-express IL-9. This data suggest that IL-9 may participate in the regulation of granulocytopoiesis in allergic inflammation.